Microbiological prospecting for petroleum is based on the theory that various hydrocarbons migrate from subterranean deposits to the surface of the earth. Such hydrocarbons are usually gases and have a marked effect on the surface of the soil. More specifically, it has been established with a considerable degree of certainty that these hydrocarbon gases feed certain microorganisms in the soil. Thus, the existence of a petroleum deposit can be recognized by either a high content of such microorganisms in the soil or by the presence in the soil of products resulting from the feeding of said microorganisms upon the hydrocarbon content of the soil. Three primary techniques have been employed: (1) gas utilization, which is the indirect measurement of the presence of hydrocarbon-consuming microorganisms by measuring the amount of a hydrocarbon consumed by such microorganisms; (2) direct measurement of the population of hydrocarbon-consuming microorganisms, e.g., by using plate counts; and (3) measurement of the metabolic products of hydrocarbon-consuming microorganisms.
The first of the above described techniques namely, gas utilization, typically involves isolating the hydrocarbon-consuming microorganisms from a soil sample and growing such microorganisms in the presence of hydrocarbon gases. By growing microorganisms isolated from soil samples in the presence of hydrocarbon gases, the type, amount and character of the resulting microbial growth are observed. This technique typically requires two weeks for its implementation and is somewhat unreliable since it wa based on subjective determinations of turbidity based on a five-point scale.
Variations of the gas-utilization technique have been made. U.S. Pat. No. 2,349,472 to Taggart describes a process wherein a soil sample is placed in a sealed chamber and the chamber filled with air and hydrocarbons. The drop in the pressure over a selected period of time indicates the bacterial content of the soil sample. Another gas utilization technique wherein pressure drops are measured is described in U.S. Pat. No. 2,665,237 to Strawinski, wherein a large excess of nutrient is used so as to ensure maximization of bacterial growth uniformly among the various samples and to render the contents of moisture and nutrient present in the sample itself inconsequential terms of the calculations carried out. The disclosures of these patents are incorporated herein by reference.
Several disadvantages have been found to exist with the gas-utilization techniques described above. A primary disadvantage is that the methods generally require several days to several weeks for completion of the tests before meaningful data or indications can be obtained. Another disadvantage is that the additional calculations based on hydrocarbon consumption by microorganisms contained in a soil sample can be very unreliable due to the highly adaptive nature of many microorganisms. It is known, for example, that microorganisms with the ability to grow on hydrocarbons are ubiquitous in nature. Therefore, although such microorganisms have the capability of utilizing hydrocarbons (or can be induced to utilize hydrocarbons upon their exposure to such compounds in the absence of their usual growth substrates), they can also metabolize organic compounds normally present in soil and not attributable to the presence of petroleum. Accordingly, measurement of the amounts of hydrocarbon-consuming microorganisms via gas consumption calculations does not necessarily mean that hydrocarbons derived from petroleum were present in the sample.
To overcome those disadvantages, it was proposed by Hitzman in U.S. Pat. No. 2,880,142, incorporated herein by reference, to subject the soil samples to the action of an organic liquid normally toxic to microorganisms, such as an alcohol, as the sole carbon and energy source for culturing the microorganisms. This method involved a direct measurement, or plate count, method (as opposed to the gas utilization methods discussed above wherein soil dilutions were placed on agar containing the alcohol and colonies were counted after a six-day incubation period. In this way, only those microorganisms which were not killed by the organic liquid and were able to utilize such organic liquid as a nutrient were counted. Thus, only the hydrocarbon-consuming microorganisms which have been consuming hydrocarbons attributable to petroleum deposits were those counted in this method. While this method offers the advantage of selectivity relative to other methods, it nonetheless has the disadvantages of the length of time required before results are available and of the laborious and time-consuming nature of counting colonies on an agar plate. Investigations subsequent to the issuance of U.S. Pat. No. 2,880,142 have revealed that not all of the microorganisms which are resistant to the organic compound and which grow on the agar plates are actually subsisting on such organic compound. To the contrary, a high proportion of such microorganisms are actually able to grow on the agar without any additional carbon source. Thus, the results obtained still may not accurately represent the number of hydrocarbon-consuming microorganisms present in the soil at the time of sampling.
In addition to the gas utilization and direct measurement, e.g., plate counting methods, the population of hydrocarbon-consuming microorganisms in a soil sample may be determined indirectly by measurements of the metabolic products of the hydrocarbon-consuming microorganisms. U.S. Pat. No. 2,269,889 to Blau, incorporated herein by reference, discloses a method which measures the bacterial oxidation products of the hydrocarbon gases. More specifically, it was believed that hydrocarbon-consuming bacteria in the soil polymerize the hydrocarbons derived from petroleum to form carboxylic acids. Thus, a chemical reagent such as sodium peroxide was employed to cause a color change by acting on the products of the microbial attack of the gases. It was further deemed possible to determine the presence of heavy organic bodies, resulting from the action of bacteria on the hydrocarbons, by examining soil samples with ultraviolet light. Oxidation-reduction potential measurements have also been found to be affected by the presence of oil deposits, presumably due to the action of bacteria on hydrocarbons. A number of shortcomings are associated with known techniques for assaying the metabolic products of hydrocarbon-consuming bacteria. Thus, as stated in U.S. Pat. No. 2,269,889, where such products are detected by a sodium peroxide-induced color change, the data collected are greatly affected by seasonal changes, the presence of calcium and magnesium in the soil, and the occurrence of any recent disturbances to the soil at the place from which the sample was taken.
From the above, it is apparent that none of the known techniques for determining the presence of hydrocarbon-consuming bacteria in a soil sample has met with great deal of success. Problems with false readings, too much subjectivity in the readings, unreasonably long time periods to complete the tests, and complexity of the analyzing techniques have pervaded this field. Not surprisingly, therefore, such techniques have gained only limited commercial acceptance.